CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells

The method presented herein is associated with the Lab Resource article titled "Generation of aMHC-EGFP knock -in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9based gene targeting " [1]. The cardiac muscle -specific protein, a -myosin heavy chain ( aMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into a MHC , the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of a MHC with a 2A non -joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining. center dot The line enables monitoring of cell maturation events during human cardiac development. center dot The line is a valuable platform for cardiotoxicity tests and drug screening. center dot This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.